While siRNAs are double-stranded oligonucleotides that perfectly pair to degrade their target mRNA, miRNAs can pair perfectly or imperfectly. Perfect base pairing leads to the degradation of the mRNA (similar to siRNA), while imperfect complementarity inhibits translation. It’s been suggested that miRNAs regulate up to 50% of all mRNAs in the human genome.

Discoveries linking miRNAs to a number of diseases have helped propel the fast-paced growth of this young field. Release of the first miRNA-based diagnostic test in 2008 helped reduce its failure risk. Keeping pace with new applications and tackling current issues was a focus of Keystone’s “MicroRNAs and Human Disease” conference held last month. Researchers described technological advances as well as novel miRNA therapeutics such as for cardiovascular disease, cancer, and muscular dystrophy.

Some of the same strategies already developed for delivery of siRNA for RNA interference (RNAi) also are being applied to miRNA. “Both miRNAs and siRNAs need to be delivered into the target tissue or cell in order to activate the desired therapeutic effect,” Muthiah Manoharan, Ph.D., senior vp, drug discovery, Alnylam Pharmaceuticals, explained.

“Chemical modifications to provide drug-like properties to RNA molecules are used in the synthesis of both siRNAs and antimicroRNAs (antimiRs). In addition, the lipid nanoparticles (LNP) platform, which has proven to be effective with siRNAs, is also showing promise with miRNAs and miRNA mimics.”

According to Dr. Manoharan, Alnylam is utilizing second-generation LNPs to enhance delivery of siRNAs in several models. “We demonstrated an approximate 100-fold improvement in potency over first-generation formulations, and achieved an effective dose (ED50) at single-digit microgram per kilogram dose levels. Additionally, we’ve made progress in hepatocyte-targeted in vivo gene silencing by modifying the siRNAs.

“We conjugated an N-acetylgalactosamine moiety (GalNac) to siRNAs and achieved improved potency using subcutaneous delivery at low, clinically relevant doses. In the case of a GalNAc-conjugated siRNA targeting transthyretin (TTR), target gene silencing was achieved with an ED50 of approximately 5 mg/kg with a single subcutaneous injection. These results represent a greater than 30-fold improvement in target gene silencing with siRNA conjugates as compared with first-generation siRNA conjugates previously described.”

Targeting Cancer

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Researchers at Regulus Therapeutics believe that miR-21 may provide a novel therapeutic target for hepatocellular carcinoma as well as other types of cancers. The company is commencing dose and mechanistic studies.

The involvement of miRNAs in cancer research emerged from studies demonstrating their aberrant expression in neoplastic tissues. Identification of several targets of miRNAs associated with cancer suggests that networks of miRNAs linked to oncogenes or tumor suppressors play pivotal roles in cancer development.

Researchers at Regulus Therapeutics described how one such miRNA (miR-21) may provide a novel therapeutic target for hepatocellular carcinoma. “Many types of cancer show an overexpression of miR-21,” noted Eric G. Marcusson, Ph.D., director of oncology.

“We worked on a mouse model that overexpresses the well-known oncogene RAS in hepatocytes. Mice develop tumors resembling those seen in humans, including expression of high levels of miR-21. We performed long-term treatment of mice with an antimiR-21 inhibitor (a single-stranded oligonucleotide) as well as a mismatched antimiR control. We found that all the control mice died while the majority of treated mice survived.”

According to Dr. Marcusson, an important aspect of these studies was the demonstration of miR-21 knockdown using genome-wide mRNA expression studies. “Such studies show specificity and target engagement, and thus, a very specific mechanism. To our knowledge this is the first demonstration of a treatment that inhibited a specific miRNA that resulted in a significant increase in survival.”

The company will now pursue dose and mechanistic studies as well as identify suitable biomarkers for clinical applications. Additionally, Zak Zimmerman, Ph.D., director of business development, says that miR-21 may also be a target for other types of cancers. “Our studies suggest not only that miR-21 is a promising therapeutic for liver cancer, but also highlight the potential of miRNA therapeutics for other tumor types and diseases of high unmet need.”

miRNAs and Heart Disease

miRNAs are also being targeted to treat cardiovascular disease. According to Eva van Rooij, Ph.D., director of biology and scientific co-founder, MiRagen Therapeutics, “Physical stress to the heart leads to pathologies such as hypertrophy, fibrosis, myocyte apoptosis, and subsequent death from pump failure and arrythmias.

“Classical pharmacological treatments such as ACE-inhibitors and beta-blockers can prolong survival of heart failure patients but ultimately cannot prevent disease progression. We found that miRNAs play a significant role in cardiovascular disease and that their modulation may provide a new therapeutic approach to its treatment.”

Dr. van Rooij’s team is targeting multiple pathological miRNAs including miR-208, a cardiac-specific miRNA. “We initially identified miRNA expression signatures in humans and mice that were associated with pathological cardiac hypertrophy, heart failure, and myocardial infarction. We next performed gain- and loss-of-function studies in mice and found profound and unexpected functions for miRNAs in numerous facets of cardiac biology.”

“One of the most remarkable findings was that while knockout mice without disease-inducing miRNAs were mostly normal, under healthy conditions they displayed an aberrant response to cardiac stress. These studies suggest that miRNAs are important regulators of disease-related processes, and that the manipulation of their expression may mitigate the effects of, or even halt, disease progression.”

Often, miRNAs regulate many different genes in many different types of tissues. “Therapeutics targeting a tissue-specific miRNA such as miR-208 present less risk for off-target effects,” Dr. van Rooij explained. “Systemic delivery of antimiRs—oligonucleotide chemistries targeting specific miRNAs—via either intravenous or subcutaneous injection is sufficient to induce potent and long lasting miRNA inhibition in cardiac tissue.”

MiRagen is in the process of performing animal studies. “We are conducting preclinical studies using antimiR approaches in rat and large animal models of heart failure to further advance our findings toward the clinic.”

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